Analysis of polynucleotides
According to some embodiments herein, methods and kits for labeling and analyzing nucleic acids are provided. In some embodiments, sequence-specific labeling is performed on polynucleotide sequences associated with a host genome, and the presence or absence of patterns characteristic of extragenomic sequences are determined.
1 . A method of determining the presence or absence of one or more foreign elements in a subject, the method comprising:
obtaining a biological sample of the subject, the biological sample comprising polynucleotides, wherein the polynucleotides comprise a genome of the subject;
removing host polynucleotides from the biological sample, wherein the host polynucleotides are bound with a methyl-CpG binding domain (MBD), and wherein the removal of the host polynucleotides comprises binding the methyl-CpG binding domain with protein A magnetic beads;
labeling the polynucleotides in a sequence-specific manner to provide a pattern of sequence-specific labels on the polynucleotides, wherein the labeling comprises non-cutting labeling of the polynucleotides, and wherein labeling the polynucleotides in the sequence-specific manner to provide the pattern of sequence-specific labels on the polynucleotides comprises labeling the polynucleotides with labeling probes or enzymes having a lower label density for the genome of the subject than for the foreign elements;
linearizing the polynucleotides in nanochannels following labeling, whereby the nanochannels comprise labeled single molecule polynucleotides;
obtaining images of labeled single-molecule polynucleotides linearized in the nanochannels;
processing the images into digital representations of relative label locations in the polynucleotides;
aligning the relative label locations on the polynucleotides in silico against a foreign reference map comprising relative locations of labels on foreign elements, thereby determining a quantity of the polynucleotides that align to relative locations of label locations of foreign elements, wherein the foreign reference map comprising relative locations of labels on foreign elements comprises a label density of about 5 labels to about 20 labels per 100 kb; and
determining the presence of foreign elements in the subject when the quantity exceeds a limit of detection, wherein the one or more foreign elements comprise a viral sequence and/or a transposable element sequence.
2 . The method of claim 1 , wherein the foreign reference map comprises relative locations of labels of at least three different foreign elements.
3 . The method of claim 1 , wherein the quantity of polynucleotides that align to relative locations of labels on foreign elements is normalized with respect to a quantity of polynucleotides aligned to a host reference map to yield an estimate of a fraction of foreign element material in the biological sample.
4 . The method of claim 1 , further comprising:
aligning the relative locations on the polynucleotides in silico against a host reference comprising relative locations of labels in a genome of the subject.
5 . The method of claim 1 , wherein the limit of detection is calculated from variability observed in multiple controls and samples containing different concentrations of host and foreign element nucleic acids.
6 . The method of claim 1 , wherein determining the presence of foreign elements in the subject comprises determining a probability that each of the foreign elements is present in the biological sample, and classifying the foreign elements as present when the probability exceeds a predefined confidence level.
7 . The method of claim 1 , wherein the labeling the polynucleotides comprises a probe having a melting temperature of 66° C. to 75° C.
8 . The method of claim 1 , wherein non-cutting labeling non-cutting label is applied by at least one of a, a non-cutting restriction enzyme, a zinc-finger protein, an antibody, a transcription methyltransferase factor, a DNA binding protein, a hairpin polyamide, a triplex-forming oligodeoxynudeotide, and a peptide nucleic acid.
9 . The method of claim 1 , wherein the non-cutting labeling is applied by a methyltransferase.
10 . The method of claim 1 , wherein the labeling the polynucleotides comprises a probe having an annealing temperature of 1° C. to 20° C. less than the melting temperature.
11 . The method of claim 1 , wherein the polynucleotides that align to relative locations of label locations of foreign elements further comprises a flanking sequence, thus identifying a site of integration of the foreign elements to a host genome.
12 . The method of claim 1 , wherein labeling comprises forming a D-loop in the polynucleotides and hybridizing at least one specific probe(s) to a first strand of the polynucleotides at the D loop with higher affinity than to a complementary strand of the first strand.